A method to investigate muscle target-specific transcriptional signatures of single motor neurons.

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Motor neurons in the vertebrate spinal cord have long served as a paradigm to study the transcriptional logic of cell type specification and differentiation. At limb levels, pool-specific transcriptional signatures first restrict innervation to only one particular muscle in the periphery, and get refined, once muscle connection has been established. Accordingly, to study the transcriptional dynamics and specificity of the system, a method for establishing muscle target-specific motor neuron transcriptomes would be required. To investigate target-specific transcriptional signatures of single motor neurons, here we combine ex-ovo retrograde axonal labeling in mid-gestation chicken embryos with manual isolation of individual fluorescent cells and Smart-seq2 single-cell RNA-sequencing. We validate our method by injecting the dorsal extensor metacarpi radialis and ventral flexor digiti quarti wing muscles and harvesting a total of 50 fluorescently labeled cells, in which we detect up to 12,000 transcribed genes. Additionally, we present visual cues and cDNA metrics predictive of sequencing success. Our method provides a unique approach to study muscle target-specific motor neuron transcriptomes at a single-cell resolution. We anticipate that our method will provide key insights into the transcriptional logic underlying motor neuron pool specialization and proper neuromuscular circuit assembly and refinement.